Figure 4.
Intracellular storage and secretion of VWF in the ECFCs as well as expression of recombinant VWF in HEK293T cells. (A) Immunofluorescence images of ECFCs isolated from the IP and healthy individuals. In healthy ECFCs, VWF (green) is stored in rod-shaped organelles, resembling WPBs. However, VWF staining in the IP-ECFCs exhibited shorter WPBs than those observed in healthy ECFCs. The scale bar is 10 µm. Boxes signify close-up views of the processed 3D models of the WPBs granules surrounding with oriented bounds, generated by arivis Vision4D 3.2. The dot plot graphs demonstrate the quantitative morphological analysis of WPBs in healthy (black dot plot) and IP-ECFCs (pale red dot plot), including the average value of the longest (length), middle (depth), shortest sides (width), and sphericity factor (Ψ) of the selected 1000 WPBs. The sphericity factor, describing the roundness of the 3D granules, is represented as a value between 0 and 1, where 1 is an ideal sphere, and any particle that is not a sphere will have a sphericity < 1. IP-ECFCs demonstrated a decrease in length (1.69 ± 0.01 µm), depth (1.08 ± 0.01 µm), and width (0.91 ± 0.01 µm) of WPBs compared with wt (with an average length, depth, and width of 2.28 ± 0.01 µm, 1.37 ± 0.01 µm, and 1.2 ± 0.01 µm, respectively; P < .001). Furthermore, IP-ECFCs WPBs displayed a slightly increased sphericity value (0.61 ± 0.00 vs 0.59 ± 0.00 of wt WPBs; P < .001), favoring a less elongated shape. (B) The graph of the mean of VWF:Ag levels in the medium of ECFCs obtained from the IP (3 independent ECFCs isolation, each 3; N = 9) and 6 healthy donors (each 3 samples; N = 18). (C) The multimer profile of VWF in the supernatant of ECFCs analyzed by electrophoresis on 1.6% and 1.2% SDS-agarose gel. The multimer of VWF secreted from IP-ECFCs exhibited loss of large and intermediate multimers along with the shift in mobility of the small multimers (red arrows). (D) VWF:Ag levels (left) and VWF:GPIb (right) of secreted VWF into the medium of the transiently transfected HEK293T with huVWF (N = 9), huVWF/huVWF Del4-34 (coexpression at a ratio of 1:1; N = 9), and huVWF Del4-34 (N = 9) as well as untransfected cells (mock). (E) The multimer of recombinant VWF secreted into the medium of transfected huVWF, cotransfected huVWF/huVWF Del 4-34, and huVWF del 4-34 in HEK293T cell lines on 1.6% SDS-agarose gel. The multimer composition of secreted coexpressed huVWF/huVWF Del4-34 demonstrates the loss of large multimers along with a shift in mobility of the small multimers (red arrows) and homozygously expressed huVWF del 4-34 exhibited no multimer as expected. Error bars indicate the SEM. NS, not significant (P > .05); ***P < .001(unpaired Student t test).

Intracellular storage and secretion of VWF in the ECFCs as well as expression of recombinant VWF in HEK293T cells. (A) Immunofluorescence images of ECFCs isolated from the IP and healthy individuals. In healthy ECFCs, VWF (green) is stored in rod-shaped organelles, resembling WPBs. However, VWF staining in the IP-ECFCs exhibited shorter WPBs than those observed in healthy ECFCs. The scale bar is 10 µm. Boxes signify close-up views of the processed 3D models of the WPBs granules surrounding with oriented bounds, generated by arivis Vision4D 3.2. The dot plot graphs demonstrate the quantitative morphological analysis of WPBs in healthy (black dot plot) and IP-ECFCs (pale red dot plot), including the average value of the longest (length), middle (depth), shortest sides (width), and sphericity factor (Ψ) of the selected 1000 WPBs. The sphericity factor, describing the roundness of the 3D granules, is represented as a value between 0 and 1, where 1 is an ideal sphere, and any particle that is not a sphere will have a sphericity < 1. IP-ECFCs demonstrated a decrease in length (1.69 ± 0.01 µm), depth (1.08 ± 0.01 µm), and width (0.91 ± 0.01 µm) of WPBs compared with wt (with an average length, depth, and width of 2.28 ± 0.01 µm, 1.37 ± 0.01 µm, and 1.2 ± 0.01 µm, respectively; P < .001). Furthermore, IP-ECFCs WPBs displayed a slightly increased sphericity value (0.61 ± 0.00 vs 0.59 ± 0.00 of wt WPBs; P < .001), favoring a less elongated shape. (B) The graph of the mean of VWF:Ag levels in the medium of ECFCs obtained from the IP (3 independent ECFCs isolation, each 3; N = 9) and 6 healthy donors (each 3 samples; N = 18). (C) The multimer profile of VWF in the supernatant of ECFCs analyzed by electrophoresis on 1.6% and 1.2% SDS-agarose gel. The multimer of VWF secreted from IP-ECFCs exhibited loss of large and intermediate multimers along with the shift in mobility of the small multimers (red arrows). (D) VWF:Ag levels (left) and VWF:GPIb (right) of secreted VWF into the medium of the transiently transfected HEK293T with huVWF (N = 9), huVWF/huVWF Del4-34 (coexpression at a ratio of 1:1; N = 9), and huVWF Del4-34 (N = 9) as well as untransfected cells (mock). (E) The multimer of recombinant VWF secreted into the medium of transfected huVWF, cotransfected huVWF/huVWF Del 4-34, and huVWF del 4-34 in HEK293T cell lines on 1.6% SDS-agarose gel. The multimer composition of secreted coexpressed huVWF/huVWF Del4-34 demonstrates the loss of large multimers along with a shift in mobility of the small multimers (red arrows) and homozygously expressed huVWF del 4-34 exhibited no multimer as expected. Error bars indicate the SEM. NS, not significant (P > .05); ***P < .001(unpaired Student t test).

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